TRPA1 is a Ca2+-permeable, non-selective cation channel and one of the key pain sensors in mammals. It has emerged as a novel target for analgesics and anti-inflammatory agents. Pain sensation mediated by TRPA1 involves modification of N-terminal cysteine residues on the channel by thiol-reactive compounds and inflammatory mediators. Binding of thiol-reactive compounds to the channel in the resting state leads to channel activation followed rapidly by desensitization. While channel activity has been studied extensively by electrophysiological methods, little is known about the structural mechanisms of channel activation and desensitization. Additionally, two compounds that directly inhibit TRPA1 are in pre-clinical trials, and understanding the mechanism of TRPA1 inhibition by these compounds will be important for advancing our knowledge of the structural differences between functionally relevant TRPA1 conformations. Using cryo-EM, site-directed mutagenesis, limited proteolysis, and mass spectrometry, in this grant we aim to establish a detailed structural understanding of the mechanisms of TRPA1 channel activation, desensitization and inhibition, which will help facilitate rational design of novel analgesics. !